DC-SIGN-Dependent Manner Viral Transfer to CD4 T Cells in a CR3

Opsonization of HIV with Complement Enhances Infection of Dendritic Cells and Viral Transfer to CD4 T Cells in a CR3 and DC-SIGN-Dependent Manner This information is current as of October 2, 2021. Hicham Bouhlal, Nicolas Chomont, Mary Réquena, Nadine Nasreddine, Héla Saidi, Jérôme Legoff, Michel D. Kazatchkine, Laurent Bélec and Hakim Hocini J Immunol 2007; 178:1086-1095; ;

doi: 10.4049/jimmunol.178.2.1086 Downloaded from http://www.jimmunol.org/content/178/2/1086

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Opsonization of HIV with Complement Enhances Infection of Dendritic Cells and Viral Transfer to CD4 T Cells in a CR3 and DC-SIGN-Dependent Manner1

Hicham Bouhlal,2* Nicolas Chomont,† Mary Re´quena,* Nadine Nasreddine,* He´la Saidi,* Je´roˆme Legoff,* Michel D. Kazatchkine,‡ Laurent Be´lec,* and Hakim Hocini*

In the present study, we demonstrated that opsonization of primary HIV-1 with human complement enhances infection of immature monocyte-derived dendritic cells (iDC) and transmission in trans of HIV to autologous CD4؉ T lymphocytes. Infection of iDC by opsonized primary R5- and X4-tropic HIV was increased 3- to 5-fold as compared with infection by the corresponding unopsonized HIV. Enhancement of infection was dependent on CR3 as demonstrated by inhibition induced by blocking Abs. The interaction of HIV with CCR5 and CXCR4 on iDC was affected by opsonization. Indeed, stromal-derived factor-1 was more Downloaded from efficient in inhibiting infection of iDC with opsonized R5-tropic HIV-1BaL (45%) than with heat-inactivated complement opsonized virus and similarly RANTES inhibited more efficiently infection of iDC with opsonized X4-tropic HIV-1NDK (42%) than with heat-inactivated complement opsonized virus. We also showed that attachment of complement-opsonized virus to DC-specific ICAM-grabbing nonintegrin (DC-SIGN) molecule on iDC and HeLa DC-SIGN؉ CR3؊ cells was 46% and 50% higher compared with heat-inactivated complement opsonized virus, respectively. Hence, Abs to DC-SIGN suppressed up to 80% and 60% the binding of opsonized virus to HeLa cells and iDC, respectively. Furthermore, Abs to DC-SIGN inhibited up to 70% of the infection of iDC and up to 65% of infection in trans of autologous lymphocytes with opsonized virus. These http://www.jimmunol.org/ results further demonstrated the role of DC-SIGN in complement opsonized virus uptake and infection. Thus, the virus uses complement to its advantage to facilitate early steps leading to infection following mucosal transmission of HIV. The Journal of Immunology, 2007, 178: 1086–1095.

ransmission of HIV-1 occurs following the passage of nism through which complement opsonized HIV leads to an in- virus through mucosal epithelial cells, i.e., cervicovaginal crease in both HIV binding to DC and virus production remains T cells upon sexual transmission (1) or intestinal cells upon unclear. postnatal transmission by breast milk (2). Semen, cervicovaginal Immature DCs (iDCs) are a key target for HIV following trans- by guest on October 2, 2021 secretions, and breast milk contain complement proteins that may epithelial passage of the virus (12, 13). It has been postulated that allow viral particles to be opsonized (3–5). Opsonization of HIV-1 certain iDCs present in the peripheral mucosa are the first immune- with complement results in enhancement of viral infection of T competent cells to encounter virus particles. Subsequent to virus and B cell lines (6, 7), primary PBMC (8), and primary monocytes/ capture by iDC, infectious HIV particles are transported to the macrophages (9). We have demonstrated that HIV particles acti- draining lymph nodes. DC differentiated from blood monocytes or vate complement in semen resulting in an ability to infect human CD34ϩ cells express CD4 and HIV-1 coreceptors CCR5 and CD4-negative epithelial cells in a complement-dependent fashion CXCR4 (14–16). Although productive infection of certain subset via CD11b/CD18 (CR3) (10). Recently, others have reported a of iDC has been controversial, iDC derived from blood monocytes crucial role of CR3 in the productive infection of dendritic cells and skin Langherans cells have been shown to be susceptible to (DC)3 by C3-opsonized HIV (11). However, the precise mecha- R5- and X4-tropic HIV in vitro (17–20). The attached HIV on DC membrane either infects target cells after interaction with CD4 and coreceptors and/or efficiently transmitted to CD4ϩ lymphocytes. *Universite´ Rene´ Descartes Paris V and Institut National de la Santeétdela DCs strongly express CR3 (98%) and other molecules that bind Recherche Me´dicale, Unite´ 743, and Equipe d’Immunite´ et Biothe´rapie Mu- queuse, Institut des Cordeliers, Paris, France; †Laboratoire d’Immunologie, Hoˆ- HIV directly through the gp120/gp41 envelope, including DC-spe- pital St. Luc, Centre de Recherches Pavillon Edouard-Asselin, Montreál, Que´bec, cific ICAM-grabbing nonintegrin (DC-SIGN) (21), which is a C- Canada; and ‡Institut National de la Santeét de la Recherche Me´dicale, Unite´681, type lectin that binds to high-mannose oligosaccharides on gp160. Paris, France DC-SIGN is involved in both the attachment of HIV to DC and the Received for publication July 29, 2005. Accepted for publication October 27, 2006. transfer of HIV-1 to CD4ϩ T cells (22). The costs of publication of this article were defrayed in part by the payment of page In the present study we assessed the role of complement recep- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. tor CR3 and that of DC-SIGN in the capture of complement-op- 1 This work was supported by Agence National de la Recherches sur le SIDA, Paris, sonized R5- and X4-tropic HIV by iDC, infection of the cells and ϩ France. transmission of HIV from iDC to autologous CD4 T cells. We 2 Address correspondence and reprint requests to Dr. Hicham Bouhlal, Institut Na- demonstrate that opsonization of virus with complement enhances tional de la Sante´ et de la Recherche Me´dicale, Unite´ 743, Equipe d’Immuniteét Biothe´rapie Muqueuse, Institut des Cordeliers, Escalier E, 2e`me Etage, 15 rue de l’Ecole de Me´decine, 75270 Paris Cedex 06, France. E-mail address: hicham. virus; HIC-OV, heat-inactivated complement-opsonized virus; SDF, stromal-derived [email protected] factor; MFI, mean fluorescence intensity. 3 Abbreviations used in this paper: DC, dendritic cell; iDC, immature DC; DC-SIGN, DC-specific ICAM-grabbing nonintegrin; AC-OV, activated complement-opsonized Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 www.jimmunol.org The Journal of Immunology 1087

the transmission of both R5- and X4-tropic HIV to T cells by facilitating the interaction of the virus with CR3 and DC-SIGN on iDC.

Materials and Methods Abs and reagents PE-conjugated anti-CCR5 (2D7), anti-CXCR4 (12G5), and PE-conjugated anti-CD4 (Leu3-a) were obtained from BD Pharmingen; PE-conjugated anti-CD11b, FITC-conjugated anti-CD11c (CR4), anti-CD35 (CR1) and anti-CD21 (CR2), endotoxin-free preparations of blocking CD11b (MO-1), blocking CD18 (7E4) and the corresponding isotypes, FITC-conjugated anti-CD83, PE-conjugated anti-CD1a, and goat anti- mouse Ab were purchased from Immunotech. Unconjugated mouse anti- DC-SIGN blocking mAbs (DC-4 and DC-6) were obtained from the AIDS Reagent Program, National Institute of Allergy and Infectious Diseases (NIAID, National Institutes of Health, Bethesda, MD). FITC- conjugated anti-DC-SIGN, anti-MHC class I HLA-DR anti-CD3, and PE-conjugated anti-CD14 are obtained from BD Pharmingen. The FITC-conjugated rabbit anti-gp120 and corresponding matched isotype were purchased from Intracell. Cultures were grown in RPMI 1640 supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin (In- vitrogen Life Technologies), and 10% heat-inactivated FCS (Dutscher). Downloaded from Recombinant human IL-2, IL-4, GM-CSF, RANTES, and stromal-derived factor (SDF)-1 were obtained from R&D Systems. Azidothymi- dine, PHA, and polyclonal anti-C3 complement fragment were obtained from Sigma-Aldrich. Normal human serum used as a complement source consisted of pooled sera of healthy individuals. Cells primary cultures and HIV-1 sources http://www.jimmunol.org/ DCs were differentiated from PBMC. After step-density gradient centrif- ugation, PBMC (107 cells/ml) were cultured in RPMI 1640/10% of normal human serum for1hat37°C. After washing, adherent cells were maintained in RPMI 1640/10% FCS, 1% antibiotics supplemented with IL-4/ GM-CSF (both at 10 ng/ml) to obtain iDC. Medium was changed every 48 h and new cytokines IL-4/GM-CSF added to the medium. Contamina- tion of iDCs with CD3ϩ T lymphocytes was Ͻ0.1% as checked by FACS. Autologous lymphocytes were obtained by stimulation of the nonadherent cell fraction of PBMC with PHA/IL-2 (2.5 ␮g/ml; 10 IU) for 72 h. Primary

X4-tropic HIV-1NDK was grown in PHA/IL-2-activated PBL. R5-tropic by guest on October 2, 2021 HIV-1BaL was ampliﬁed in macrophages cultures. Virus particles were quantiﬁed by measuring p24 HIV protein (HIV-p24 ELISA; DuPont). Tro- pism of viruses was determined using U87 cells positive for CD4 and CCR5 or CXCR4, obtained from the AIDS Reagent Program, NIAID, Na-

tional Institutes of Health. The TCID50 of each strain was measured and virus stored at Ϫ80°C. Cell staining Cells (0.5 ϫ 106 cells per assay) were collected in cold PBS/0.01% sodium azide/0.5% BSA, washed and incubated with conjugated monoclonal or isotype-matched Abs for 30 min at 4°C. Following a washing step in PBS/ 0.01% sodium azide, cells were ﬁxed with 1% paraformaldehyde, and 5000 events were analyzed using a FACSCalibur and the CellQuest software (BD Biosciences). Opsonization of HIV-1 and infection of iDC Both R5 and X4 HIV-1 particles (1 ng/ml p24), corresponding to 5 ϫ 102

TCID50, were opsonized by activated complement-opsonized virus (AC- OV) by adding a similar volume of normal human serum for1hat37°C, 2ϩ containing veronal buffer (0.6 mM CaCl2 and 0.9 mM MgCl2 (VBS )). As negative control, HIV-1 particles were incubated with heat-inactivated complement-opsonized virus (HIC-OV) serum and in some experiments with medium (unopsonized virus).

Treated HIV-1NDK or HIV-1BaL (1 ng/ml HIV p24) were then incubated with iDC (5 ϫ 105 cells) for3hat37°C. In some experiments, cells were incubated, with endotoxin-free preparations of blocking CR3 Abs (7E4 for CD18; MO-1 for CD11b used separately or in combination at 10 ␮g/ml),

FIGURE 1. Phenotypic analysis of differentiation of iDC by flow cy- tometry. A, At day 6, cells were collected in PBS/azidothymidine 0.01%/ receptors expressed on iDC at day 6. B, Dot plot of CD11b, CD11c, CD35, BSA 0.5% and incubated with mAb directed against CD14, HLA-DR, and CD21 expression. The mean percentage of positive cells (%) calculated CD83, CD1a or anti-CCR5, CXCR4, CD3, CD4, and DC-SIGN. Data are from ten separate experiments is indicated in corresponding quadrant. FL-1 expressed as the percentage (%) of positive cells for one representative and FL-2 correspond to the isotype Ab conjugated to the corresponding outcome of five separate experiments. Phenotypic analysis of complement fluorochrome (FITC or PE). 1088 COMPLEMENT-DEPENDENT HIV INFECTION OF DC VIA CR3 AND DC-SIGN Downloaded from

FIGURE 2. Infection of iDCs by primary R5-tropic HIV-1BaL and X4- http://www.jimmunol.org/ tropic HIV-1NDK. A, Cells were inoculated with HIV for 3 h and washed, FIGURE 3. Enhancement of iDC infection by complement opsonized and medium was added. Supernatants were collected every 48 h, and p24 by activated complement-opsonized virus (AC-OV) HIVBaL and HIVNDK. levels were measured. Results are expressed as the mean Ϯ SD of p24 con- heat-inactivated complement opsonized virus (HIC-OV) and unopsonised centration in four separate experiments. B, Total DNA was extracted for 6 h virus (UV). A, The p24 level was determined in cultured supernatants every and 6 days after infection of iDCs with 1 ng (lanes 1, 3, 5, and 7)and5ng 48 h. Results are expressed as the mean Ϯ SD of p24 concentration from four

(lanes 2, 4, 6, and 8)ofHIVBaL and HIVNDK p24 Ag. A semiquantitative PCR separate experiments. Statistical significance was calculated for AC-OV and of the HIV pol gene was conducted on purified DNA. As control, the ubiq- HIC-OV p24 produced at day 6, using the unpaired Student t test and consid- p Ͻ 0.01. B, Total DNA ,ءء ;p Ͻ 0.01 ,ء .uitous ␤-globin gene was amplified by PCR in parallel experiments. ered significant at a value of p Ͻ 0.05 was extracted from 6-h postinfected iDCs with the unopsonised virus, HIC-

OV, and AC-OV HIVBaL and HIVNDK (1 ng of p24) and a semiquantitative by guest on October 2, 2021 with human recombinant chemokines SDF-1 and RANTES (2.5 ␮g/ml), PCR of the HIV pol gene was conducted. As control, the ubiquitous ␤-globin with Abs anti-DC-SIGN (10 ␮g/ml), for 30 min at room temperature prior gene was ampliﬁed by PCR in parallel experiments. to infection. The cells were intensively washed with RPMI 1640 to elim- inate unbound virus, serum and blocking Abs excess, and cultured in fresh RPMI 1640 containing FCS for 6 days in absence of additional cytokines ϫ 5 or Abs. Residual HIV p24 released by iDC in the culture medium has been cells (5 10 cells) were incubated with opsonized or unopsonized HIV- estimated after inhibition of viral replication by azidothymidine molecule 1NDK (10 ng/ml HIV p24) for1hat37°C. Cells were washed with RPMI ␮ at 5 ␮M. Culture supernatants were collected every 48 h and viral content 1640 to remove unbound virus and lysed by adding 200 l of PBS-Triton monitored by means of HIV-1 p24 ELISA. In coculture assays, iDCs were X-100 at 0.5%, before quantiﬁcation of HIV p24 Ag. In some experiments, incubated with virus for1hat37°C, and washed before the addition of cells were incubated with a combination of DC-SIGN-blocking mAbs ␮ autologous IL-2-stimulated lymphocytes at a 1:5 ratio of iDC to lympho- (DC-4/DC-6) at 10 g/ml and polyclonal anti-C3 complement component ␮ cytes. Cocultures were maintained in the presence of IL-2 for 6 days. (10 g/ml) before addition of virus. The DC-SIGN-negative HeLa cells were used to evaluate the DC-SIGN-independent HIV binding. In addi- Detection of HIV-1 DNA in infected iDC tional experiments, iDC (106 cells) were preincubated with DC-SIGN- blocking mAbs (DC-4/DC-6) or blocking CD11b mAb (MO-1) for 30 min

Amounts of HIV-1 DNA in iDC were quantified at 6 h and 6 days fol- at room temperature before adding HIC-OV and AC-OV HIV-1BaL (10 lowing infection with opsonized or unopsonized HIV used at 1 and 5 ng/ml ng/ml HIV p24) for1hat37°C. Cells were then washed and incubated p24 Ag. DNA was extracted from washed cellular pellets using the Qiagen with FITC-conjugated anti-gp120 and PE-conjugated anti-CD1a mAbs QIAamp DNA mini kit, according to the manufacturer’s instructions. A for1hat4°C. Following a washing step in PBS/0.01% sodium azide, semiquantitative PCR of the pol gene was conducted as previously de- cells were fixed with 1% PFA and 5000 events were analyzed using a scribed (23). The PCR was performed with the oligonucleotides P63 (5Ј- FACSCalibur and the CellQuest software. HIV binding levels were ex- GCC ATT TAA AAA TCT GAA AAC AGG-3Ј) and P58 (5Ј-GAC AAA pressed as gp120 stained cells. CTC CCA CTC AGG AAT CCA-3Ј) for 37 cycles (94°C for 30 s, 53°C for 30 s, and 72°C for 60 s) in a reaction volume of 50 ␮l, containing Milli-Q Statistical analysis ␮ water, DNA templates, reaction buffer (1.5 mM MgCl2, 200 M each dNTP, 250 nM of each primer, and 2.5 U of TaqDNA polymerase) pro- Quantitative analysis is expressed as the mean and SE. An unpaired Student’s vided by the manufacturer (Promega). To check for the quality of extracted t test was used to determine the statistical significance of the data and a value Ͻ DNA and the lack of PCR inhibitors, the ubiquitous ␤-globin gene was for p 0.05 was considered as the level of statistical significance. amplified in parallel experiments by PCR, as described (24). The final PCR products were visualized under UV transillumination by means of ethidium Results bromide staining after electrophoresis in 2% agarose. Immature DCs are susceptible to infection with both primary Attachment of HIV to DC-SIGN on HeLa cells and iDC R5- and X4-tropic HIV-1 At the time of collection on day 6 of culture, most nonadherent The HIV-nonpermissive HeLa cells that stably express DC-SIGN molecule Ϫ (90–95%) were used to evaluate the HIV DC-SIGN-dependent attachment mononuclear cells exhibited an iDC phenotype (CD14 , HLA- ϩ ϩ in absence of complement receptors, HIV receptors, and coreceptors. HeLa DR , CD1a ) expressing low levels of CD83. The iDC expressed The Journal of Immunology 1089 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 4. Inhibition by CR3 blocking Abs of complement-dependent infection of iDC and transmission in trans to autologous CD4ϩ T lymphocytes. Cells were preincubated with a combination of CD11b and CD18 blocking Abs (mAb anti-CR3) or matched isotypes all used at 10 ␮g/ml, before addition of AC-OV and HIC-OV. Immature DC infected with HIVBaL (A) and HIVNDK (B) iDC infected with HIVBaL and HIVNDK and cocultured with IL-2- activated autologous lymphocytes at a 1:5 ratio (iDC to T cell). The viral production was quantified by p24 production in cell supernatants at day 6. Results are presented as the mean Ϯ SD of p24 concentrations from five separate experiments. Statistical significance was calculated for p24 production at day 6 in the presence and absence of anti-CR3 Abs, using the unpaired Student t test and considered significant at a value of p Ͻ 0.05.

CD4 at 70 Ϯ 20%, CCR5 at 20 Ϯ 10%, CXCR4 at 30 Ϯ 15%, and and/or internalized in the absence of infection, cells were cultured up to 80 Ϯ 10% DC-SIGN molecule (Fig. 1A). The iDC expressed in the presence of 5 ␮M azidothymidine, an inhibitor of the viral high levels in mean ﬂuorescence intensity (MFI) of both CR3 retro transcription step. The results showed that Ͻ2% of input (98%, 700–800 MFI) and CR4 (80%, 250–300 MFI). Only 0.2% virus (1 ng/ml p24) corresponding to 20 pg/ml HIV p24 was de- and 20% of the cells expressed CR2 and CR1, respectively (Fig. tected in iDC-infected cultures. Infection of iDC, by HIV was 1B). HIV infection of iDC was observed with both X4-tropic further conﬁrmed by nested PCR on HIV pol DNA at 6 h and at

(HIV-1NDK) and R5-tropic (HIV-1BaL) strains (Fig. 2A). A plateau day 6 postinfection (Fig. 2B). of p24 Ag production in cultures was reached at day 6 after infection. At the peak of infection (day 6) with HIV-1 and HIV- CR3-dependent enhancement of iDC infection by BaL opsonized HIV-1 1NDK, the amounts of HIV p24 Ag released in culture supernatants of 5 ϫ 105 cells were of 2500 Ϯ 560 and 500 Ϯ 55 pg/ml, re- Complement opsonization of HIV-1 particles resulted in a 3- to spectively. To evaluate the amount of HIV particles adsorbed 5–fold increase in HIV p24 production in culture supernatants at 1090 COMPLEMENT-DEPENDENT HIV INFECTION OF DC VIA CR3 AND DC-SIGN Downloaded from

FIGURE 5. Inhibitory effect of SDF-1 and RANTES chemokines on infection of iDC with complement-opsonized virus. SDF-1 (A) and RANTES (B)at2.5␮g were preincubated with iDC for 30 min at room tem- http://www.jimmunol.org/ perature before infection by AC-OV or HIC-OV of

HIV-1BaL and HIV-1NDK (1 ng/ml p24). Viral production was quantified at day 6 postinfection by HIV p24 ELISA. Results are presented as the mean Ϯ SD of p24 concentrations calculated in picograms per milliliter from five separate experiments. Statistical significance was calculated using the unpaired Student t test and p Ͻ ,ء .considered significant for values of p Ͻ 0.05 .p Ͻ 0.02 ;ءء ,0.03 by guest on October 2, 2021

day 6 postinfection as compared with production observed in the unopsonized virus (Fig. 3B). The role of complement receptor CR3 absence of opsonization of HIV (Fig. 3A). No enhancement of in facilitation of iDC infection with complement-opsonized virus infection was observed when viruses had been incubated with was investigated by incubating the cells with anti-CR3 (MO-1, 10 HIC-OV. Higher amounts of HIV DNA were present in cells that ␮g/ml) for 1 h before infection. Cells were cultured in the ab- had been infected with opsonized HIV-1BaL and HIV-1NDK as sence of additional cytokines or Abs, and HIV p24 concentra- compared with cells infected with unopsonized virus. HIV DNA tion was determined at day 6 postinfection. Anti-CR3 blocking levels were similar in experiments using HIC-OV and those using Ab suppressed up to 85% of infection with AC-OV HIV-1BaL The Journal of Immunology 1091 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 6. Implication of DC-SIGN in infection of iDC and in infection in trans of autologous lymphocytes. A, At day 6, cultured iDCs were preincubated with mAbs to DC-SIGN (DC-4/DC-6) or matched isotype for 30 min at room temperature before infection with AC-OV and HIC-OV. B,In similar conditions as in A with addition of IL-2-activated autologous lymphocytes at a 1:5 ratio. The virus was quantified by HIV p24 ELISA. Results are presented as the mean Ϯ SD of p24 concentrations calculated from five separate experiments. Statistical significance was calculated using the unpaired .p Ͻ 0.02 ,ءء ;p Ͻ 0.03 ,ء .Student t test and considered significant at a value of p Ͻ 0.05

and HIV-1NDK (6500 vs 1500 pg/ml for HIV-1BaL and 1900 vs Opsonization of HIV-1 with complement modulates the usage

700 pg/ml HIV p24 for HIV-1NDK in the absence and presence by HIV of CCR5 and CXCR4 of blocking Abs, respectively) (Fig. 4A). The role of CD11b and The effect of complement opsonization on the use of the corecep- ␤ ␣ CD18, and chains forming CR3, in the amplification of iDC tors CCR5 and CXCR4 by HIV was investigated by incubating infection with complement-opsonized virus has also been in- iDC with RANTES and SDF-1 before the addition of either vestigated. We found that the anti-CD11b (MO-1) used alone AC-OV or HIC-OV. Cells were cultured in absence of additional achieves 60–85% of inhibition, whereas anti-CD18 (7E4) in- cytokines, and p24 concentration was determined by ELISA at day hibits Ͻ20% the infection with AC-OV (data not shown). In 6 after infection. Our results showed that SDF-1 inhibited more following inhibition experiments, only a combination of both efficiently infection of iDC with AC-OV R5-tropic HIV-1BaL (53% Abs (MO-1 plus 7E4; 10 ␮g/ml each) was used. When autolo- of inhibition) than with the HIC-OV (Ͻ1%) ( p Ͻ 0.03). Similarly, gous IL-2-activated lymphocytes were added (in 1:5 ratio) to RANTES inhibited at 56% and Ͻ5% infection of iDC with Ͻ iDC that had been incubated with anti-CR3 Abs before be in- AC-OV and HIC-OV X4-tropic HIV-1NDK, respectively ( p fected, we observed an inhibition of 65% and 60% of the trans- 0.02). In contrast, SDF-1 was less potent in inhibition of iDC in- mission in trans of AC-OV HIV-1BaL and HIV-1NDK to CD4 fection with AC-OV X4-tropic strain HIV-1NDK (51%), than cells, respectively. No significant inhibition was observed when HIC-OV (90%) ( p Ͻ 0.02%). RANTES inhibited equally infec-

CR3-blocking Abs were used with HIC-OV HIV-1BaL and HIV- tion of iDC with AC-OV and HIC-OV R5-tropic strain HIV-1BaL

1NDK (Fig. 4B). Isotype-matched Ab controls had no effect on (80% of inhibition vs 78% for HIC-OV) (Fig. 5B). Preincubation infection and transmission in trans of HIV. of iDC with monoclonal anti-CD4 Ab inhibited infection with 1092 COMPLEMENT-DEPENDENT HIV INFECTION OF DC VIA CR3 AND DC-SIGN

FIGURE 7. Attachment of AC- OV and HIC-OV to HeLa DC- SIGN-positive cells and to iDC. A, HeLa DC-SIGN-positive and -negative cells (5 ϫ 105 cells) were incubated with AC-OV and HIC-OV

HIV-1NDK (10 ng of HIV p24) for 1 h at 37°C. Cells were washed and lyzed, before quantification of HIV p24 Ag. In some experiments, cells were incubated with mAbs anti-DC- Downloaded from SIGN (DC-4/DC-6) and polyclonal anti-C3 before addition of virus. Re- sults are presented as the mean Ϯ SD of p24 concentrations from three separate experiments. Statistical significance was calculated using the unpaired Student t test and consid- http://www.jimmunol.org/ ered significant when p Ͻ 0.05. B, iDCs (106 cells) were preincubated with blocking mAbs anti-DC-SIGN (DC-4/DC-6) and mAb anti-CR3 (MO-1) for 30 min at room temperature before adding HIC-OV and AC-OV HIV-1BaL (10 ng/ml HIV p24) for1hat37°C. Bind- ing of HIV to iDC was determined by guest on October 2, 2021 by HIV-gp120 staining using the FITC anti-gp120 Abs, and matched isotype was used as negative control. The percentage is the mean of positive cells (%) calculated from four separate experiments as indicated in corresponding quadrant.

AC-OV and HIC-OV X4-tropic and R5-tropic viruses at a similar the presence of AC-OV and HIC-OV. Preincubating with anti-DC- degree (80%) (data not shown). SIGN Abs (DC-4 and DC-6 at 10 ␮g/ml each) resulted in a greater

inhibition of infection of iDC with AC-OV R5-tropic HIV-1BaL Complement opsonization enhances the transmission of HIV ϩ (55%) than in the case of the corresponding HIC-OV (10%). A from iDC to CD4 T lymphocytes through increased similar result was observed when infection was conducted with attachment to DC-SIGN AC-OV X4-tropic HIV-1NDK (65%) as compared with the corre- Complement opsonization of HIV resulted in a 2- to 3-fold en- sponding HIC-opsonized virus (15%) ( p Ͻ 0.02) (Fig. 6A). We hancement of infection in trans of T lymphocytes when these cells further found that anti-DC-SIGN Abs induced a significantly were added to iDCs that had been infected with AC-OV compared higher inhibition of transmission of AC-OV R5-tropic HIV-1BaL with HIC-OV. We examined the role of DC-SIGN in infection of (60%) and X4-tropic HIV-1NDK (70%) to autologous T cells than iDC and in their ability to transmit virus to autologous T cells in of HIC-OV HIV-1BAL (58%) and HIV-1NDK (46%) (Fig. 6B). To The Journal of Immunology 1093 highlight the mechanism by which complement-opsonized HIV tivation through a direct interaction with C1q and/or C3 (25–28). infects more efficiently iDC, we have investigated the role of DC- Activation of complement results in the deposition of C3 frag- SIGN using a transfected epithelial HeLa cell line that stably ex- ments on the viral surface without efficient formation of the C5b-9 press DC-SIGN molecule but does not express CR1, CR2, CR3, terminal complex and without lysis of the virus because of the and CR4 complement receptors. As shown in the Fig. 7A, HIV presence of complement regulatory proteins on the viral membrane complement opsonization enhances up to 50% of the attachment of (27–29). Opsonized virus can divert the complement system to its ϩ virus to DC-SIGN Hela cells as compared with HIC-OV. The advantage by using the CR3/CR4 receptor to infect iDC and other attachment of complement-opsonized HIV occurred through complement receptor-expressing cells (30). Furthermore, binding DC-SIGN as demonstrated by the 80% of inhibition induced by of complement-opsonized HIV to CR3 may induce specific sig- Abs directed to DC-SIGN. Furthermore, polyclonal Abs directed naling cascades in iDCs, which are favorable for HIV replication. against C3 complement fragment induced also an inhibition of We further investigated the role of opsonization of HIV in facili- 60% of attachment of opsonized virus (Fig. 7A). As control the tating the transmission in trans of virus from iDC to autologous binding of HIC-OV and AC-OV to DC-SIGN-negative HeLa cells CD4ϩ T cells. We observed that opsonization of HIV enhanced by represented Ͻ5% of specific attachment observed with HeLa DC- 2- to 3-fold viral transfer to CD4ϩ T cells that do not express CR3 SIGN-positive cells. The role of DC-SIGN molecule has been also molecule. Enhancement of viral transmission was in part depen- investigated using iDC incubated with HIC-OV and AC-OV. Our dent on CR3 on iDC because anti-CR3 mAbs inhibited by ϳ60% results showed that the positive cells stained by FITC-conjugated transmission in trans of opsonized HIV-1BaL and HIV-1NDK.Itis anti-gp120 Abs increased from 20% to 66% (corresponding to known that Abs directed against CR3 (MO-1 plus 7E4) exert a 70% of HIV binding enhancement) when HIC-OV and AC-OV

blocking effect on CR3 function and binding of C3 components, in Downloaded from were used, respectively (Fig. 7B). The anti-DC-SIGN and anti- the absence of cross-linking by a secondary Ab. The role of CD11b CR3 blocking Abs decreased the binding of AC-OV from 66% to and CD18, ␤ and ␣ chains forming CR3, in the infection in trans 28% (58% of inhibition) and to 32% (50% of inhibition), respec- of C4 T cells with complement-opsonized virus has been investi- tively. In contrast, the binding of HIC-OV (20%) was not signif- gated separately. To avoid any interference of anti-CR3 Abs with icantly changed in the presence of Abs anti-DC-SIGN and anti- receptors expressed on T cells, iDCs that had been preincubated CR3. As control, matched isotype Abs achieved Ͻ5% of inhibition

with MO-1 and 7E4 were washed before being added to target http://www.jimmunol.org/ of HIV attachment. Down modulation of expression of iDC marker CD4 T cells. We found that the anti-CD11b (MO-1) used alone CD1a in the presence of HIV and complement fragment is prob- achieves 60% of inhibition, whereas anti-CD18 (7E4) inhibits ably due to the maturation priming of immature DC. Ͻ10% the infection with AC-OV (data not shown). This effect is Discussion very specific because no significant inhibition was observed when CR3-blocking Abs were used with HIC-OV HIV-1 In the present study, we demonstrate that opsonization of HIV with BaL and HIV-1 . complement enhances both the infection of iDC and the transmis- NDK Because mucosal secretions contain active complement compo- sion in trans of virus to CD4 T lymphocytes. Enhancement of nents (5, 31) one may speculate that virus released in the mucosal infection of iDC observed following preincubation of virus with lumen becomes opsonized and acquires an enhanced potential for by guest on October 2, 2021 normal serum was complement-dependent and occurred with both attachment to and infection of iDC, a key target cell for HIV in the R5- and X4-tropic primary isolates of HIV-1. Thus, productive early steps of sexual transmission of the virus (12). Furthermore, com- infection of iDC was seen much earlier and was 5-fold higher with AC-OV than in the presence of corresponding HIC-OV and un- plement opsonized virus already attached to iDC, may interact with CR1 complement receptor expressed on CD4 T lymphocytes induc- opsonized virus. The virus produced in supernatant at day 6 was ϩ infectious as demonstrated by productive infection of IL-2-acti- ing an enhancement of infection in trans of autologous CD4 T lym- vated lymphocytes. In addition, proviral HIV-1 DNA was detected phocytes (7). Interestingly, complement receptors were also shown to in iDC within6hofincubation with complement-opsonized play a role in the capture and docking of virus on the surface of HIV-1, the amounts of HIV-1 pol DNA measured by semiquanti- uninfected follicular DCs in germinal centers (32, 33). However, tative nested PCR being significantly higher in cells infected with CR2 rather than CR1 and CR3, was shown to be the main binding opsonized HIV, as compared with cells infected with virus that had molecule for opsonized HIV on follicular DCs (34). been preincubated with culture medium. These data extend previ- We further investigated the role of CD4, CXCR4, CCR5, and ous observations on the enhancing effect of complement on infec- DC-SIGN in the infection of iDC with unopsonized and comple- tion of monocytes/macrophages (8, 9) and more recently of CD4- ment-opsonized HIV. We have shown that the iDC express CD4, negative, complement receptor-positive epithelial cells (10). The CCR5, and CXCR4 and are susceptible to the productive infection role of complement receptor (CR3) was shown by the blocking by both R5- and X4-tropic strains. This result is comforted by the effect of anti-CR3 mAbs on infection of iDC with opsonized HIV- recent observation demonstrating that X4-tropic strains replicate in iDC (20). We demonstrate for the first time, that complement op- 1BaL and HIV-1NDK. The fact that anti-CD11b and anti-CD18 mAbs inhibited 70–80% and Ͻ20%, respectively, infection of sonization of HIV modulates the inhibitory activity of RANTES ␣ and SDF-1 on infection of iDC with X4- and R5-tropic viruses. iDC by opsonized virus, is in favor of implication of M integrin ␤ Indeed, SDF-1 was significantly more efficient in inhibiting infec- and 2 chains (CR3) in interaction of opsonized virus with iDC membrane. However, the opsonized virus may also interact with tion of iDC with the opsonized R5-tropic HIV-1BAL (45%) than the CR4 molecules that are also expressed on iDC (250–300 MFI with the corresponding unopsonized virus (Ͻ1%). Similarly, for CD11c compared with 700–800 MFI for CD11b). Investigat- RANTES was more efficient in inhibiting infection of iDC with ing this situation is complicated by the fact that no neutralizing opsonized X4-tropic HIV-1NDK (42%) than with the unopsonized Abs against CR4 receptor are available. In contrast, the anti- virus (Ͻ5%). Furthermore, HIV complement opsonization reduced CD11a (LFA-1) Abs inhibited Ͻ10% of infection with opsonized significantly the effect of TAK-779, a CCR5 antagonist molecule, virus. Several regions in gp120/gp41 including the conserved C2 on infection of DCs by CCR5-tropic HIV (data not shown). region, the carboxyl-terminal flank of V3 loop and the transmem- The modulation of virus tropism by complement opsonization is brane gp41 domain have been shown to support complement ac- probably not the result of an alteration of coreceptors expression 1094 COMPLEMENT-DEPENDENT HIV INFECTION OF DC VIA CR3 AND DC-SIGN consequently to the binding of complement fragments to DCs. In- DC-SIGN but does not express CR1, CR2, CR3, and CR4 com- deed, we have previously shown that the membrane expression of plement receptors. We demonstrated that AC-OV attached up to CD4, CCR5, and CXCR4 was not affected by the presence of 2-fold greater than HIC-OV and unopsonized virus. The attach- complement components (9). Our results, both on epithelial cells ment of complement-opsonized HIV to HeLa cells occurred and DCs, are in favor of an effect of opsonins on the interactions through DC-SIGN as demonstrated by 80% inhibition of attach- between gp160 and coreceptors. Our results suggest that covalent ment observed in the presence of Ab to DC-SIGN. Furthermore, interaction between major viral glycoprotein (gp120/gp41) and the polyclonal Ab directed against C3 fragment of complement inhib- C3 fragments (iC3b) induces a modification in interaction of HIV ited at 58% attachment of opsonized virus to HeLa DC-SIGN cells. and coreceptors used in viral entry into iDC. Three major sites on The role of DC-SIGN molecule was also confirmed using iDC. gp120 are able to activate the complement system: the second Indeed, the binding of complement opsonized HIV to iDC was constant region of gp120 C2 (233–251), the third constant region decreased by 50% in the presence of anti-DC-SIGN Abs. These of gp120 and the third variable region V3 loop. These regions are results are in favor of a direct interaction between complement able to activate complement in the absence of Abs (26). Interest- fragments and DC-SIGN molecule. Hence, the better interaction of ingly, The variable V3 loop is also responsible for viral tropism DC-SIGN with complement opsonized HIV gp120 results in a determination allowing an interaction with one of two major HIV higher ability of virus to attach to HeLa DC-SIGNϩ cells and to coreceptors CCR5 and CXCR4. Thus, it is implied that V3 loop iDC. HIV complement activation may also occur through the lectin and C2-C3 in interaction with iC3b probably modulate the core- pathway where virus binds mannose-binding lectin present in se- ceptors recognition on target cell membrane. In contrast, blocking rum. One may suggest that mannose-binding lectin bind to HIV mAb directed to CD4 receptor did not exhibit a differential effect gp120 before interaction with DC-SIGN expressed on DCs. Inter- on inhibition of infection with AC-OV and HIC-OV X4-tropic estingly, it has been reported that mannose-binding lectin acts as Downloaded from

HIV-1NDK and R5-tropic HIV-1BAL. This result agrees with the recognition molecules for infectious agents that colonize the cer- observation that the CD4 binding site on gp120 does not implicate vicovaginal mucosa, suggesting that the lectin pathway of com- complement-activating sites such as C2, V2, C5, and V3 loop (35). plement activation may play a crucial role in HIV transmission in Opsonization of HIV by complement probably enhances the bind- female genital tract (37). ing to CR3 receptor and increases the number of viral particles Taken together, our results indicate that complement opsoniza- adsorbed on cells allowing a more efficient fusion and viral entry tion of viral particles affects the interactions of HIV with DC- http://www.jimmunol.org/ through CD4 and HIV coreceptors. All these observations indicate SIGN, CCR5, and CXCR4. HIV-1 would thus turn complement that opsonization with complement alters the phenotype of HIV opsonization to its own replicative advantage, resulting in en- and transform it from a strict R5- and X4-tropic to a dual tropic hanced infection of iDC and transmission to CD4 T lymphocytes. virus that allows HIV to use both CCR5 and CXCR4 coreceptors. In summary, we demonstrate that complement-opsonized HIV ac- The latter observation is not in favor of a selection process of quires a dual tropism for CCR5- and CXCR4-expressing cells and R5-tropic HIV during the early phases of mucosal penetration of the ability to attach to and infect iDC through complement recep- HIV, suggesting that it occurs at later stages of the infection. In- tors (CR3) and DC-SIGN-dependent pathways. Thus, the virus terestingly, as for iDC, the inhibitory effect of RANTES was in- probably uses active complement in secretions to facilitate early by guest on October 2, 2021 creased in the case of infection of CD4-negative HT-29 epithelial steps leading to infection following mucosal transmission of HIV. cells with opsonized X4-tropic HIV-1 (10). However, the inhibitory effect of SDF-1 was decreased in the case of both opsonized Acknowledgments X4- and R5-tropic viruses, suggesting that opsonized virus may We thank Morgan Klein for participation in infection experiments. The not interact in a similar fashion with CCR5 and CXCR4 on epi- following reagent was obtained through the National Institutes of Health thelial cells and iDC. AIDS Research and Reference Reagent Program, Division of AIDS, Na- During the early steps of HIV transmission, iDC and CD4ϩ T tional Institute of Allergy and Infectious Diseases, National Institutes of Health: Unconjugated mouse mAbs to DC-SIGN (clone DC-4, catalog no. lymphocytes synergize in facilitating initial viral replication. Im- 5442; clone DC-6, catalog no. 6456; U87 CCR5, catalog no. 4035; and mature DCs capture and transmit virus to CD4 T lymphocytes U87 CXCR4, catalog no. 4036) are from Dr. F. Baribaud. leading to a greater sensitivity to the infection of these cells (34– 36). HIV-free particles attach to iDC through several molecular Disclosures pathways, including C-type lectin receptors, e.g., the DC-SIGN The authors have no financial conflict of interest. molecule that is well expressed by DCs of human genital mucosae (36). It is now admitted that DC-SIGN contributes to the capture References virus responsible for transmission to T cells shortly after viral up- 1. 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